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Approximately 25-50 million Americans, 30 million Europeans, and 8% of the Aus-tralian population have a rare disease. Rare diseases are thus a common problem for clini-cians and account for enormous healthcare costs worldwide due to the difficulty of establish-ing a specific diagnosis. In this article, we review the milestones achieved in our understanding of rare diseases since the emergence of next-generation sequencing (NGS) technologies and analyze how these advances have influenced research and diagnosis. The first half of this review describes how NGS has changed diagnostic workflows and provided an unprecedent-ed, simple way of discovering novel disease-associated genes. We focus particularly on meta-bolic and neurodevelopmental disorders. NGS has enabled cheap and rapid genetic diagnosis, highlighted the relevance of mosaic and de novo mutations, brought to light the wide pheno-typic spectrum of most genes, detected digenic inheritance or the presence of more than one rare disease in the same patient, and paved the way for promising new therapies. In the sec-ond part of the review, we look at the limitations and challenges of NGS, including determina-tion of variant causality, the loss of variants in coding and non-coding regions, and the detec-tion of somatic mosaicism variants and epigenetic mutations, and discuss how these can be overcome in the near future.

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Hyperinsulinism of Infancy (HI) is a clinical disorder characterized by deregulation of insulin secretion that leads to profound hypoglycemia. Mutations in genes encodingthe ATP-regulated potassium channels of the pancreatic β-cell, namely ABCC8 (SUR1) and KCNJ11 (Kir6.2), are the major genetic known cause of the disease. To elucidate the genetic etiology of HI in the uncharacterized Spanish population, we conducted extensive sequencing analysis of the ABCC8 (83.5Kb) and KCNJ11 (1.7Kb) genes in 34 Spanish HI patients. Mutations in ABCC8 were detected for both alleles in 13 patients, while ten patients carried only one mutation in one of the ABCC8 alleles. We have detected 22 novel and seven previously described mutations in ABCC8, ∼60% of them lead to a premature termination signal, which would result in truncated SUR1 proteins. No mutations were found in the KCNJ11 gene. In addition, we report for the first time a 3914bp macrodeletion associated with the HI disorder. The potential pathogenicity of several additional variants is discussed. The spatial pattern of three pathological mutations suggests possible geographical founder effects. This work reveals for first time the involvement of KATP channels in the pathogenesis of an important proportion (∼68%) of Spanish HI patients. The spectrum of mutations in Spanish HI patients provides an important tool for diagnosis and prognosis of HI patients in the Spanish population, as well as for genetic counseling of HI families. © 2006 Wiley-Liss, Inc.KEY WORDS: hyperinsulinism; ABCC8; KCNJ11; mutation spectra; Spanish INTRODUCTIONATP-sensitive potassium channels (KATP) are major regulators of glucose-induced insulin secretion in pancreatic ß cells . Mutations in ABCC8 (MIM# 600509) and KCNJ11 (MIM# 600937) genes have been shown to result in defects in the KATP channel function leading to Hyperinsulinism of Infancy HI, a disease characterized by deregulated insulin secretion that leads to severe and recurrent hypoglycaemias DOI: 10.1002/humu.9401 2 Fernández-Marmiesse et al. ; references therein). Estimates for the incidence of this disorder vary from 1/40.000 live births in Northern Europe (Bruining et al. 1990) to 1/2675 live births in Saudi Arabia (Mathew et al. 1988). The severity of the disease varies from a mild form, which responds to medical treatment, to severe drug-resistant form, which may need resection of the pancreas. Early diagnosis is important to avoid irreversible brain damage due to profound hypoglycaemia. Clinical manifestations of HI, which occurs predominantly in neonates and infants, include large birth weight for gestational age, seizures, cyanosis and coma (Hussain et al. 2000).Although both autosomal dominant and recessive inheritance of HI have been described, this disorder is inherited as an autosomal recessive trait in the majority of familial cases. Most of HI cases (>95%) are sporadic, and two histopathologic lesions can be distinguished, a diffuse and a focal form, each with a prevalence of ∼50% . It has been demonstrated that focal ca...

BackgroundWith over 50 different disorders and a combined incidence of up to 1/3000 births, lysosomal storage diseases (LSDs) constitute a major public health problem and place an enormous burden on affected individuals and their families. Many factors make LSD diagnosis difficult, including phenotype and penetrance variability, shared signs and symptoms, and problems inherent to biochemical diagnosis. Developing a powerful diagnostic tool could mitigate the protracted diagnostic process for these families, lead to better outcomes for current and proposed therapies, and provide the basis for more appropriate genetic counseling.MethodsWe have designed a targeted resequencing assay for the simultaneous testing of 57 lysosomal genes, using in-solution capture as the enrichment method and two different sequencing platforms. A total of 84 patients with high to moderate-or low suspicion index for LSD were enrolled in different centers in Spain and Portugal, including 18 positive controls.ResultsWe correctly diagnosed 18 positive blinded controls, provided genetic diagnosis to 25 potential LSD patients, and ended with 18 diagnostic odysseys.ConclusionWe report the assessment of a next–generation-sequencing-based approach as an accessory tool in the diagnosis of LSDs, a group of disorders which have overlapping clinical profiles and genetic heterogeneity. We have also identified and quantified the strengths and limitations of next generation sequencing (NGS) technology applied to diagnosis.

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BackgroundIn patients with phenylketonuria (PKU), a low-phenylalanine (Phe) diet supplemented with low-protein foods and a Phe-free amino acid mixture favors a dietary intake rich in carbohydrates, but little is known about how these molecules are metabolized in this setting. The objective of the present study was to analyze carbohydrate metabolism in patients with hyperphenylalaninemia.MethodsWe conducted a multicenter cross-sectional study to investigate biochemical markers of basal and postprandial carbohydrate metabolism in PKU patients according to age, Phe tolerance, waist circumference and body mass index (BMI), diet, tetrahydrobiopterin (BH4) supplementation, and adherence to treatment. Basal biomarkers and anthropometric parameters were also evaluated in patients with mild hyperphenylalaninemia (MHPA) and in healthy controls.ResultsA total of 83 patients aged 4–52 years were studied; 68.7% had PKU and 31.3% had MHPA. 68 healthy controls of similar sex and age were also evaluated Metabolic control was adequate in 71.9% of PKU patients. Fasting glucose levels (mean 80.77 ± 8.06 mg/dL) were high in just one patient, but fasting insulin levels, with a mean of 12.74 ± 8.4 mIU/L, were altered in 15 PKU patients (26.3%) and markedly higher than in patients with MPHA (p = 0.035). Fasting insulin levels and Homeostasis Model Assessment Insulin Resistance (HOMA-IR) were significantly higher than in healthy controls and correlated with body mass index, waist circumference, age, and also showed statistically significant differences according to diagnosis and Phe tolerance (p < 0.05). Patients under BH4 therapy had lower insulin levels and HOMA-IR. A higher mean carbohydrate intake from AA mixtures was observed in classic PKU patients. The caloric intake in the form of carbohydrates was also higher in PKU than MHPA patients (p = 0.038) and it was correlated with basal insulin (rho = 0.468, p = 0.006), HOMA-IR (rho = 0.423, p = 0.02), BMI (rho 0.533, p = 0.002), and waist circumference (rho 0.584, p = 0.0007).ConclusionsThis study shows that PKU patients are at risk of carbohydrate intolerance and insulin resistance, more evident in adults and overweight patients, probably related to their higher caloric intake in form carbohydrate content. A higher dependency of AA mixtures was demonstrated in PKU patients.Electronic supplementary materialThe online version of this article (10.1186/s13023-018-0847-x) contains supplementary material, which is available to authorized users.

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